Soluble steroidal peptides for nucleic acid delivery

ABSTRACT

Amphiphilic lipopeptide compositions for gene delivery are disclosed. An illustrative amphiphilic lipopeptide composition includes a human protamine 2 peptide conjugated to a hydrophobic moiety. Illustrative hydrophobic moieties include sterols, bile acids, and fatty acids. The amphiphilic lipopeptide composition is mixed with a nucleic acid such that the nucleic acid binds to the peptide portion of the lipopeptide. This mixture is placed in contact with mammalian cells to effect transfection of the cells with the nucleic acid. A method of making such amphiphilic lipopeptides is also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/302,725, filed Jul. 3, 2001, which is hereby incorporated byreference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

BACKGROUND OF THE INVENTION

This invention relates to gene delivery. More particularly, thisinvention relates to compositions of matter and methods of use andmaking thereof for gene delivery wherein the compositions of mattercomprise amphiphilic lipoproteins configured for binding nucleic acids.

Progress in the area of gene delivery has been tremendous in the lastseveral years, yet in clinical settings the dream of a successfultherapy based on nucleic acids remains a frustrating riddle. Genedelivery vectors have been largely put into either of two categories,viral or non-viral, and most of the published reports have focused oncircumventing the deficiencies inherent in both types of vectors.Factors such as toxicity, permanently altering the host genome throughrecombination events with viral vectors, and poorly optimized deliverycapabilities with non-viral vectors demand application of concepts thathave clinical relevance in terms of safety, efficacy, and patientcompliance. However, by now its clear that feasibility of a singlevector serving as a universal gene carrier for all disease targets isremote and impractical.

In cancer immunotherapy, for example, the use of cytokines such asinterleukin-12, which is a proven anti-proliferative cytokine inconfinement and inhibition of tumor progression and metastasis inseveral types of cancers in vivo, is of great interest. Althoughcytokine gene therapy has been attempted with a variety of viralvectors, such as adenovirus, retroviris, adeno-associated viruses, andlentiviruses, there is still a growing need to optimize non-viral genecarriers with unprecedented safety and efficacy profiles. Among existingnon-viral gene carriers polyethyleneimine, and lipid-protamine-DNA (LPD)lipoplexes have had some success in terms of cytokine gene transferefficiency, however the issues related to carrier-associated toxicitiesare poorly understood. Cationic lipids are water insoluble and requirethe formation of liposomes using a colipid, such as dioleylphophatidylethanolamine (DOPE) or cholesterol in presence of organicsolvents, which involves multiple steps. Although their gene transferapplications have been under investigation since 1987, the exactmechanism elucidating their structure-function relationship has not beencompletely revealed. It is believed that lipid anchors, such as steroidsand fatty acid chains, serve to provide amphiphilic character to thesecarriers, which would orient the head group surface charge morefavorably, and also take part in hydrophobic interactions with plasmaand organelle membranes. Lipid anchors can also interact specificallywith various membrane receptors for enhanced cellular uptake andlipid-mediated transduction. Polyethyleneimine and Starburst™dendrimers, due to their high transfection efficiency, have received alot of attention and remain, by far, the most effective cationicpolymers for transfection created to date. The functioning of thesepolymers has been attributed to the so-called proton sponge effect dueto secondary and tertiary amines present in these polymers, whichsupposedly leads to disruption inside endosomes and endo-lysosomes byosmotic swelling. Gene carriers that would combine the concepts of watersolubility, amphiphilic nature, lipid mediated membrane interactions,endosomal buffering, and nuclear targeting would be an exciting optionfor plasmid based gene therapy.

Peptide based gene delivery systems are least investigated, and theirapplications in delivering cytokine genes are virtually unexplored.Several different types of peptides that possess endosomolytic,fusogenic, or membrane permeabilizing properties derived from variousviruses, such as vesicular stomatitis virus glycoprotein (VSVG) andinfluenza virus hemaglutinin, have been used either alone or incombination with liposomes and polymers. Co-polymers of lysine andhistidine have also been shown to efficiently deliver genes insidecells, and this property has been attributed to the imidazole ring ofthe histidine side chain, which behaves as an endosomal rupturing agent.

While prior compositions and methods for delivering peptides are knownand are generally suitable for their limited purposes, they possesscertain inherent deficiencies that detract from their overall utility.For example, polyethyleneimine is effective only in high molecularweight (>10,000 M.W.) formulations, but such high molecular weightcompositions can be toxic, elicit immune responses, arenon-biodegradable, are not site specific, and condense plasmid DNA tootightly. Cationic lipids can be toxic at therapeutic doses, elicitimmune responses, require several steps to synthesize and involve theuse of organic solvents, are water insoluble, offer little inherentendosomal buffering, and are not site specific. Current peptide-basedgene carriers may be toxic at therapeutic doses, elicit immuneresponses, often require cationic lipids for effectiveness, frequentlyare subject to aggregation, and exhibit poor water solubility due tohydrophobic amino acid residues.

In view of the foregoing, it will be appreciated that providingcompositions and methods for delivering peptides, especially cytokines,would be a significant advancement in the art.

BRIEF SUMMARY OF THE INVENTION

These and other objects can be addressed by providing a compositioncomprising a PRM2 peptide conjugated to a hydrophobic moiety. In oneillustrative embodiment of the invention, the PRM2 peptide comprises apeptide identified herein as SEQ ID NO:2.

The hydrophobic moiety illustratively comprises a sterol, a bile acid,or a fatty acid. Illustrative sterols include cholestanol, coprostanol,cholesterol, epicholesterol, ergosterol, and ergocalciferol. Examples ofbile acids include cholic acid, deoxycholic acid, chenodeoxycholic acid,lithocholic acid, ursocholic acid, ursodeoxycholic acid,isoursodeoxycholic acid, lagodeoxycholic acid, glycocholic acid,taurocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid,dehydrocholic acid, hyocholic acid, and hyodeoxycholic acid. Examples offatty acids include C₄-C₂₀ alkanoic acids, such as butyric acid, valericacid, caproic acid, caprylic acid, capric acid, lauric acid, myristicacid, palmitic acid, and stearic acid.

In another illustrative embodiment of the invention, the PRM2 peptide isconjugated to the hydrophobic moiety through an amide (peptide) linkage.Other linkers known in the art may also be used according to theinvention. The hydrophobic moiety can be conjugated to the PRM2 peptidethrough a non-terminal amino acid residue, thus forming a “T-shaped”conjugate.

Another illustrative embodiment of the invention comprises a mixture ofa nucleic acid and a conjugate comprising a PRM2 peptide and ahydrophobic moiety. The nucleic acid binds to the PRM2 peptide portionof the conjugate. The nucleic acid can be a plasmid or other type ofnucleic acid known in the art for gene delivery.

Still another illustrative embodiment of the invention comprises amethod for transfecting a mammalian cell, such as a human cell,comprising contacting the cell with a composition comprising a mixtureof a nucleic acid and a conjugate comprising a PRM2 peptide and ahydrophobic moiety, and then incubating the cell under conditionssuitable for growth thereof.

Yet another illustrative embodiment of the invention comprises a plasmidconfigured for expressing p35 and p40 subunits of interleukin-12 undercontrol of at least one cytomegalovirus promoter. An illustrativeconfiguration of this plasmid is p2CMVmIL-12.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 shows a schematic representation of an amphiphilic lipopeptideaccording to the present invention.

FIG. 2 shows a schematic representation of synthesis of an illustrativesoluble steroidal peptide according to the present invention.

FIG. 3 shows a mass spectrum of the illustrative soluble steroidalpeptide of FIG. 2; a major peak is at 2315.22, which corresponds to themolecular weight of this soluble steroidal peptide, and minor peaks areevident at 2272.13, 2332.08, and 2394.96, which account for <5%impurities.

FIGS. 4A-B show membrane permeabilization of cultured CT-26 cells withthe soluble steroidal peptide of FIG. 2 in the presence of ethidiumbromide. FIG. 4A shows permeabilization as a function of pH, wherein aspH 5 and 6 less than 25% staining was observed. FIG. 4B showspermeabilization as a function of concentration of the soluble steroidalpeptide.

FIG. 5 shows a circular dichroism spectrum of the soluble steroidalpeptide of FIG. 2.

FIG. 6 shows viability of CT-26 colon adenocarcinoma cells aftertransfection with SSP/p2CMVmIL-12 complexes prepared at various N/Pratios in 5% (w/v) glucose and 0.25 M phosphate buffer, pH 8.0. Nakedp2CMVmIL-12 and SuperFect/p2CMVmIL-12 (9.23/1, N/P) were used forcomparison. Relative cell viability was at least 80% for allSSP/p2CMVmIL-12 complexes with N/P ratios ≦50/1. In contrast,SuperFect/p2CMVmIL-12 complexes upon transfection resulted in less than60% cell viability.

FIG. 7 shows luciferase activity in cultured cells as well as CT-26subcutaneous tumors 48 hours after transfection. Non-transfected cellswere used as negative controls. Luciferase activity is expressed asRLU/mg of total protein.

FIG. 8 shows ELISA for mIL-12 levels in cultured CT-26 colonadenocarcinoma cells. Non-transfected cells were used as negativecontrols.

DETAILED DESCRIPTION

Before the present compositions of matter, methods of making thereof,and methods of use thereof are disclosed and described, it is to beunderstood that this invention is not limited to the particularconfigurations, process steps, and materials disclosed herein as suchconfigurations, process steps, and materials may vary somewhat. It isalso to be understood that the terminology employed herein is used forthe purpose of describing particular embodiments only and is notintended to be limiting since the scope of the present invention will belimited only by the appended claims and equivalents thereof.

The publications and other reference materials referred to herein todescribe the background of the invention and to provide additionaldetail regarding its practice are hereby incorporated by reference. Thereferences discussed herein are provided solely for their disclosureprior to the filing date of the present application. Nothing herein isto be construed as an admission that the inventors are not entitled toantedate such disclosure by virtue of prior invention.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to a composition comprising a mixture of “a conjugate” and “anucleic acid” includes a mixture of two or more of such conjugatesand/or two or more of such nucleic acids.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outbelow.

As used herein, “comprising,” “including,” “containing,” “characterizedby,” and grammatical equivalents thereof are inclusive or open-endedterms that do not exclude additional, unrecited elements or methodsteps. “Comprising” is to be interpreted as including the morerestrictive terms “consisting of” and “consisting essentially of.”

As used herein, “consisting of” and grammatical equivalents thereofexclude any element, step, or ingredient not specified in the claim.

As used herein, “consisting essentially of” and grammatical equivalentsthereof limit the scope of a claim to the specified materials or stepsand those that do not materially affect the basic and novelcharacteristic or characteristics of the claimed invention.

As used herein, “peptide” means peptides of any length and includesproteins.

As used herein, “PRM2 peptide” means a peptide derivative of humanprotamine 2 and configured for binding to a nucleic acid. For example,amino acid residues 51-63 of human protamine 2 comprise the sequenceidentified herein as SEQ ID NO:1, which is rich in arginine andhistidine residues, and binds to nucleic acids. In a PRM2 peptideaccording to the present invention, the cysteine residue of SEQ ID NO:1was replaced with a lysine residue, and an additional histidine residuewas added at both the amino and carboxy termini of SEQ ID NO:1 to resultin the peptide identified as SEQ ID NO:2. The lysine residue provides anε-amino residue for linkage to the hydrophobic moiety.

Thus, illustrative PRM2 peptides include the peptide having the aminoacid sequence identified as SEQ ID NO:1 and biologically functionalequivalents thereof, such as peptides having the amino acid sequenceidentified herein as SEQ ID NO:2. Such functional equivalents retainfunctionality in binding nucleic acids, although they may betruncations, deletion variants, or substitution variants of SEQ ID NO:1or include additional amino acid residues attached thereto.

As mentioned above, changes may be made in the structure of the PRM2peptide while maintaining the desirable nucleic acid-bindingcharacteristics. For example, certain amino acid residues may besubstituted for other amino acid residues in a protein structure withoutappreciable loss of interactive binding capacity with structures suchas, i.e., antigen-binding regions of antibodies or binding sites ofligands such as an IL-2 receptor-binding peptide. Since it is theinteractive capacity and nature of a protein that defines that protein'sbiological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence and nevertheless obtaina protein with like properties. It is thus contemplated that variouschanges may be made in the amino acid sequence of a PRM2 peptide withoutappreciable loss of its biological utility or activity.

It is also well understood by the skilled artisan that inherent in thedefinition of a biologically functional equivalent protein or peptide isthe concept that there is a limit to the number of changes that may bemade within a defined portion of the molecule and still result in amolecule with an acceptable level of equivalent biological activity. Itis also well understood that where certain residues are shown to beparticularly important to the biological or structural properties of aprotein or peptide, e.g. residues in active sites, such residues may notgenerally be exchanged.

Amino acid substitutions are generally based on the relative similarityof the amino acid side-chains relative to, for example, theirhydrophobicity, hydrophilicity, charge, size, and the like. An analysisof the size, shape, and type of the amino acid side-chains reveals, forexample, that arginine, lysine, and histidine are all positively chargedresidues; that alanine, glycine, and serine are all a similar size; andthat phenylalanine, tryptophan, and tyrosine all have a generallysimilar shape. Therefore, based upon these considerations, the followingconservative substitution groups or biologically functional equivalentshave been defined: (a) Cys; (b) Phe, Trp, Tyr, (c) Gln, Glu, Asn, Asp;(d) His, Lys, Arg; (e) Ala, Gly, Pro, Ser, Thr, and (f) Met, Ile, Leu,Val. M. Dayhoff et al., Atlas of Protein Sequence and Structure (Nat'lBiomed. Res. Found., Washington, D.C., 1978).

To effect more quantitative changes, the hydropathic index of aminoacids may be considered. Each amino acid has been assigned a hydropathicindex on the basis of its hydrophobicity and charge characteristics,which are as follows: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine (+2.5); methionine (+1.9); alanine(+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan(−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate(−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine(−3.9); and arginine (4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is generally understood inthe art. J. Kyte & R. Doolittle, A simple method for displaying thehydropathic character of a protein, 157 J. Mol. Biol. 105-132 (1982). Itis known that certain amino acids may be substituted for other aminoacids having a similar hydropathic index or score and still retain asimilar biological activity. In making changes based on the hydropathicindex, the substitution of amino acids whose hydropathic indices arewithin ±2 is desired, within ±1 is particularly desired, and within ±0.5is even more particularly desired.

It is also understood that an amino acid can be substituted for anotherhaving a similar hydrophilicity value and still obtain a biologicallyequivalent protein. As detailed in U.S. Pat. No. 4,554,101, thefollowing hydrophilicity values have been assigned to amino acidresidues: arginine (+3.0); lysine (+3.0); aspartate (+3.0); glutamate(+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine(0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine(−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine(−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5);tryptophan (−3.4).

In making changes based upon similar hydrophilicity values, thesubstitution of amino acids whose hydrophilicity values are within ±2 isdesired, within ±1 is particularly desired, and within ±0.5 is even moreparticularly desired.

As used herein, “hydrophobic moiety” means a hydrophobic entity that canbe conjugated to a hydrophilic peptide for obtaining an amphiphiliccomposition. Illustrative hydrophobic moieties according to the presentinvention include steroids, such as sterols and bile acids, and fattyacids. Illustrative sterols that can be used in the present invention,either as is or after activation to permit more facile conjugation to ahydrophilic peptide, include cholestanol coprostanol, cholesterol,epicholesterol, ergosterol, ergocalciferol, and the like. Illustrativebile acids that can be used in the present invention, either as is orafter activation to permit more facile conjugation to a hydrophilicpeptide, include cholic acid, deoxycholic acid, chenodeoxycholic acid,lithocholic acid, ursocholic acid, ursodeoxycholic acid,isoursodeoxycholic acid, lagodeoxycholic acid, glycocholic acid,taurocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid,dehydrocholic acid, hyocholic acid, hyodeoxycholic acid, and the like.Illustrative fatty acids that can be used in the present invention,either as is or after activation to permit more facile conjugation to ahydrophilic peptide, include C4 to C20 alkanoic acids, such as butyricacid, valeric acid, caproic acid, caprylic acid, capric acid, lauricacid, myristic acid, palmitic acid, stearic acid, and the like.

FIG. 1 shows a schematic representation of an amphiphilic lipopeptideaccording to the present invention. The amphiphilic lipopeptide includesa hydrophilic peptide head group, represented by A, which is conjugatedto a hydrophobic entity, represented by B. The hydrophilic peptide iscoupled to the hydrophobic entity by a linker, represented by L, whichcan be any of a variety of linkers known in the art for linking chemicalsubunits together into a whole unit, such amide linkages, includingpeptide linkages, urethane linkages, disulfide linkages, ether linkages,and the like. The amino- and carboxy-termini of the hydrophilic peptideare represented by N and C, respectively. Illustratively, thehydrophobic entity is linked to the hydrophilic peptide through anon-terminal amino acid residue, represented by M, thus conferring onthe amphiphilic lipopeptide a “T-shaped” configuration. FIG. 1 alsoshows that the hydrophobic entity can be linked through certain moietiesthereof. For example, in the case where the hydrophobic entity is asteroid, the C₁-C₅ end of the steroid, represented by X, may beillustratively linked to the peptide, and the C₂₂-C₂₇ end of thesteroid, represented by Y, is not directly linked to the peptide. Otherconfigurations are also possible within the scope of the presentinvention, however, as is exemplified in the examples below. By way offurther example, in the case where the hydrophobic entity is a fattyacid, the carboxylic acid end of the fatty acid, represented by X, islinked to the peptide, and the carbon chain end of the fatty acid,represented by Y, is not directly linked to the peptide.

EXAMPLE 1 Synthesis, Purification, and Physicochemical Characterization

The hydrophilic peptide head group of an illustrative amphiphiliclipopeptide, SSP, is based on the peptide sequenceHis-Tyr-Arg-Arg-Arg-His-Cys-Ser-Arg-Arg-Arg-Leu-His (SEQ ID NO:1)corresponding to amino acid residues 51-63 of human protamine 2 (PRM2),which is rich in arginine and histidines. The cysteine residue at aminoacid 57 amino acid was replaced with lysine for providing an ε-aminogroup for linkage to a hydrophobic moiety, and an additional histidineresidue was included on both the N-terminus and the C-terminus to yieldthe peptide chainHis-His-Tyr-Arg-Arg-Arg-His-Lys-Ser-Arg-Arg-Arg-Leu-His-His (SEQ IDNO:2). The entire synthesis scheme is shown in FIG. 2.

The peptideH₂N-His-His-Tyr-Arg-Arg-Arg-His-Lys-Ser-Arg-Arg-Arg-Leu-His-His-COOH(SEQ ID NO:2) was synthesized by solid phase method on rink amide resinusing the standard 9-fluorenylmethoxycarbonyl (Fmoc) strategy on anApplied Biosystems 433A peptide synthesizer.2,2,4,6,7-Pentamethyl-dihydrobenzofurane-5-sulfonyl (Pbf), Trityl (Trt)and 1-(4,4-dimethyl-2,6-dioxocyclohexylidene) 3-methylbutyl (ivDde) wereused as protective groups for arginine, histidine and lysine sidechains, respectively, and for other amino acids, tertiary-butoxycarbonyl(t-Boc) was used as the protecting group. After synthesis, the peptideattached to the resin was briefly treated with 5% trifluoroacetic acidto remove the Dde protective group from the ε-amine of lysine sidechains. It was then washed multiple times with 100% methanol to obtainapproximately 50 μM of peptide for further reactions. Activated steroidwas prepared as follows: 30.105 mg ofO—(N-Succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TSTU,Aldrich, Milwaukee, Wis.) was dissolved in 100 μl of dimethyl formamide(DMF), 174.2 μl of pure diisopropyl ethylamine (DIPEA, Sigma ChemicalCo., St. Louis, Mo.) was dissolved in 825.8 μl of DMF, and 376.58 mg oflithocholic acid (LA, Aldrich, Milwaukee, Wis.) was dissolved in 1 mlDMF to obtain their 1 Molar solutions. The TSTU solution (100 μl) wasgradually dispensed in 100 μl of LA solution in the presence of 3 foldmolar excess of DIPEA and then allowed to react for a period of 2 hoursat room temperature with mild shaking. The reaction mixture was thenallowed to react with the peptide in a total reaction volume of 2 ml for12 hours at room temperature with shaking. The reaction mixture waswashed several times with DMF followed by deprotection with 95%trifluoroacetic acid (TFA, Aldrich, Milwaukee, Wis.) for a period of 90minutes at room temperature. The supernatant solution was separated fromthe resin after centrifugation at 1000 g. The final reaction yieldwas >80% with respect to starting amounts of peptide. The deprotectedsteroidal peptide was purified using reverse phase HPLC on a Vydac C18column (4.6×250 mm with 2×20 guard). The purified soluble steroidalpeptide (SSP) was >93% pure and its composition was confirmed by aminoacid analysis. The trifluoroacetic acid attached to the SSP was replacedby acetic acid and lyophilized until further use. SSP was characterizedusing Matrix Assisted Laser Desorption Ionization Time Of Flight(MALDI-TOF) mass spectrometry (m/z=2534.12; FIG. 3). The concentrationof SSP was determined by using ε_(274.5 mm) of 1400 M⁻¹cm⁻¹ forun-ionized tyrosine side chain and was reconstituted in water at aconcentration of 10 mg/ml until further use.

EXAMPLE 2

The procedure of Example 1 was followed except that 413.7 mg ofcholesteryl carboxylic acid (CA) was substituted for lithocholic acid.

Cholest-5-ene-3β-carboxylic acid (CCA) was synthesized as follows. Asolution of methyl magnesium iodide was prepared under reflux by pouring25 ml tetrahydrofuran (THF) on 400 mg Mg turnings with a pinch of iodineand a few drops of CH₃I in a 3-neck flask for 10 min. After the vigorousreaction subsided, a solution of 4 g 3β-chlorocholest-5-ene in 50 ml THFwas added drop wise over a period of 3 hours. After refluxing for 3hours, the reaction was cooled to room temperature. To this mixture waspoured finely ground dry ice (10 g), followed by stirring for 1 hour.This solution was cooled in an ice bath and hydrolyzed by adding icecold 1 M H₂SO₄ (100 ml). After stirring for 5 min, 10 g NaCl and 100 mldiethyl ether were added. The layers were separated, and then theaqueous layer (bottom layer) was again extracted with diethyl ether (100ml). The combined organic (ethereal) layers were washed 5 times with asolution of Na₂S₂O₃.5H₂O (120 mg) in H₂O (30 ml) to remove a persistentorange color. Finally the organic layer was washed with double-distilledH₂O (30 ml) several times. The organic layer was filtered to remove thesuspension of bicholesteryl. The filtrate was then dried with MgSO₄ andagain filtered. The organic layer was concentrated in vacuum and vacuumdried for 18 hours. The crude solid was triturated with ice-cold hexanes(3×50 ml). The solid was vacuum dried for 1 h. The product obtained (0.5g, 12.5% yield) was ˜90% pure and was used for further reactions assuch.

EXAMPLE 3 Membrane Permeabilization Study

Membrane permeabilization studies were performed to find out whether SSPwas able to form pores through plasma membranes and whether thisphenomenon had any pH dependence or not. SSP prepared according toExample 1 was used at micromolar concentrations on cells preincubatedwith ethidium bromide, which intercalates between the strands of dsDNAand causes it to fluoresce. Ethidium bromide on its own is a poormembrane permeant, however, if the membranes are permeabilized, it wouldrapidly cross the cytoplasm, accumulate in the nucleus, and bind to thecellular DNA. Its fluorescent property would allow us to thus find theextent to which permeabilization via SSP occurred.

The membrane permeabilization activity of SSP was determined using flowcytometry. CT-26 cells were grown overnight and at 70-80% confluencywere trypsinized and centrifuged. Cells were washed several times in1×PBS (pH 7.4) and eventually resuspended in it to make cell suspensionsof 1×10⁶ cells/ml. One microliter of ethidium bromide (10 mg/ml) wasmixed in 0.5 ml of cell suspension aliquots and stored at 4° C. 15minutes before use. The pH of cell suspensions was adjusted to values5,6,7, or 8 using either 1N NaOH or 1N HCl solutions 5 minutes beforestarting the experiment. SSP was finally used to permeabilize themembranes at the same final concentration for pH studies or at differentconcentrations at pH 7.4. Temperature effects at 4° C. and 37° C. werealso studied to find any temperature dependent differences inpermeabilization. After 5 and 15 minutes incubation, the cellfluorescence intensity was measured using FACScan Analyzer (BectonDickinson, Sunnyvale, Calif.), which has a single 15-mW argon (488 nm)laser light source. A 585 nm long pass filter was used to collect theemitted red fluorescence. The forward scatter, side scatter, andethidium bromide fluorescence were simultaneously recorded with 10,000cells at 300 events per second. The results were later analyzed usingcell quest software provided by the same manufacturer. Histogramstatistics were used to quantitatively determine the extent ofpermeabilization.

FIG. 4A shows the pH dependence and the time dependence of the extent ofpermeabilization when 10 μM SSP was used at pHs 5, 6, 7 and 8. At pHsabove the pKa of the imidazole ring of histidine (pKa=6.1), there waspoor fluorescence and very few cells (<25%) were stained. However, atlower pHs, such as 5 and 6, the cells were stained rapidly, within aperiod of 5 minutes, and over 97% cells were stained. FIG. 4B shows thefluorescent staining of genome at different concentrations of SSP. Highlevels of staining (>85%) were achieved when cells were incubated with 5μM of SSP or above, and almost 100% staining levels were achieved at 10μM SSP and above. These results indicate that SSP facilitates rapiddiffusion in a concentration dependent fashion, which occurs only at pHswhere imidazole ring of histidine is completely deprotonated.

EXAMPLE 4 Secondary Structure of SSP

It was desirable to find out whether SSP showed any secondary structuresin solutions with different pHs and, if so, to determine the pattern ofchange with decreasing values of pH. Circular dichroism (CD)measurements were performed at 25° C. at pH 5, 6, 7, and 8 using an Aviv62DS spectrometer (Aviv Associates, Lakewood, N.J.) and a 0.1 cmpathlength quartz cuvette. SSP concentration used in each case was 25μM. Wavelength scans at 1 nm bandwidth, 1 nm per step with 5 s dataaveraging for each sample were repeated 4 times and later, averaged andcorrected by buffer baseline spectrum. Peptide alone was also used ascontrol to distinguish between the SSP and peptide and any likelycontributions by steroid anchor.

FIG. 5 shows the circular dichroism wavelength scans from 195 nm to 280nm for SSP at various pHs, and, as expected, SSP did not show anysecondary structure. For comparisons, the same peptide sequence (SEQ IDNO:2) was used in control experiments to account for any structuralchanges induced by the steroid anchor. However, no significant secondarystructure formation was found. This could be explained by the fact thatSSP contained mainly arginines and several histidines. Positivelycharged arginines would have mutual electrostatic repulsion and wouldproject an extended conformation in solution, which would retain SSP asa T-shape molecule. This type of structure proves two things. First, themembrane permeabilization could be mainly due to deprotonated histidineresidues, and there was no contribution due to any changes in thesecondary conformations of SSP such as change in the α-helical structurewhich is reminiscent of the way some viral peptide sequences work topermeabilize plasma membranes and endosomal compartments. Second, theextended sequence of SSP would provide maximum exposure of arginines tonegatively charged phosphodiester bonds thus minimizing the amount ofSSP needed to condense the pDNA into small particles. T-shaped cationicamphiphiles have also been shown to facilitate much higher geneexpression compared to conventional I-shaped amphiphiles.

EXAMPLE 5 Condensation with DNA, Particle Size, and Zeta Potential

SSP was characterized in terms of condensation with plasmid DNA,particle size, and zeta potential.

IL-12 is a heterodimeric cytokine encoded by two separate genes, p40 andp35. It is naturally produced by macrophages and B lymphocytes. Plasmidp2CMVmIL-12 was constructed with each subunit-encoding gene under thetranscriptional control of a separate cytomegalovirus (CMV) promoter,which has been described previously. Briefly, EcoRI and XmaI restrictionenzyme sites were introduced into the p35 and p40 cDNAs by polymerasechain reaction using pCAGGS-IL-12 as a template. After PCR reactions,the p35 and p40 cDNAs were purified by 1% agarose gel electrophoresisand electroelution. The purified cDNAs were digested with EcoRI and XmaIand inserted into pCI plasmid (Promega, Madison, Wis.), resulting inconstruction of pCMV-p35 and pCMV-p40, respectively. The expression unitof p40 in pCMV-p40 was then isolated by digestion with Bg1 II and BamHI,followed by 1% agarose gel electrophoresis. The isolated p40 expressionunit was inserted at the BamHI site of pCMV-p35. The resulting plasmid,p2CMVmIL-12 was confirmed by restriction enzyme assays. A plasmidencoding luciferase under control of a simian virus 40 (SV40) promoter,pGL3-control vector, hereinafter, “pLuc”) was used as a reporter geneand was purchased from Promega (Madison, Wis.). Plasmids were amplifiedusing the DH5α strain of E. coli (Promega, Madison, Wis.) and purifiedusing EndoFree™ Qiagen Kit (Qiagen, Boulder, Colo.) following themanufacturer's protocols. Plasmids were characterized by UVspectrophotometric assay at 260/280 nm and 0.7% agarose gelelectrophoresis to determine the purity, integrity and concentration ofthe plasmids; restriction enzyme assay to confirm that there was norearrangement of the genes during cloning and propagation. The opticaldensity ratios at 260 mm to 280 nm of these plasmid preparations were inthe range of about 1.7 to 1.8.

Various formulations of SSP/p2CMVmIL-12 were prepared as follows: 100 μlaliquots of SSP at various concentrations and pDNA (0.2 mg/ml) wereprepared in 4.5% (w/v) glucose to bring the osmolality to 285-295 mOsm,and 1M phosphate buffer was used to bring the pH to 8.0 of bothaliquots. SSP/pDNA complexes at various N/P ratios ranging from 0.5/1(N/P) to 50/1 (N/P) were prepared by rapidly mixing contents of SSPaliquot in pDNA aliquots using a pipettor. N/P ratio was calculated bytaking into account 3 nitrogens for each arginine in SSP, therebyresulting in 18 nitrogens for each SSP molecule. The SSP/pDNA complexeswere incubated for 15 minutes at room temperature beforeelectrophoresis.

ζ-potential and particle size of PAGA/plasmid complexes were measured asfollows: SSP/pDNA complexes were prepared and then diluted in 3.8 ml of0.1 μm filtered water to bring the volume to 4 ml. The samples weresubjected to mean particle size measurement using ZetaPALS (BrookhavenInstruments Corp, Holtsville, N.Y.). Following the determination ofparticle size, the samples were evaluated for their electrophoreticmobility by the same equipment using the same light source andwavelength. All experiments were performed at 25° C. and 677 nmwavelength at a constant angle of 15°. The ζ potential was automaticallycalculated from the electrophoretic mobility based on Smoluchowski'sequation. The particle size was reported as effective mean diameter.

SSP condensed pDNA completely at N/P ratios 3/1 and above under theaforementioned formulation conditions. The SSP/pDNA complexes resultedin small particle size in the range of 136 nm to 56 nm for N/P ratiosranging from 5/1 to 50/1. The guanidinium group of arginine remainsprotonated across pH range 1-13 and provides efficient pDNA condensationin strongly buffered solutions. The SSP/pDNA complexes were stable forabout 1-2 weeks and did not show any signs of aggregation or change insurface charge which was confirmed by a repeat of particle sizing, zetapotential, and gel electrophoresis. It is envisaged that small sizeSSP/pDNA complexes would enable higher levels of intratumoraldispersibility after local administration as well as efficient cellularuptake through endocytosis.

The exact mechanisms governing nucleocytoplasmic trafficking are notclearly known, however, it is likely that after events such as cellularuptake, endocytosis and yet unknown mechanisms the SSP/pDNA complexeswill remain in cytoplasm before nuclear translocation of pDNA. SSP andpDNA alone were also tested for their cytosolic stability. To study thestability of SSP/p2CMVmIL-12 complexes inside cytoplasm, these complexeswere incubated them with cytosolic extract and the results indicate thatpDNA was stable for long periods of time when complexed with SSP. Theproblems encountered during pDNA transfer are greatly influenced bycytosolic nucleases and SSP was able to protect the pDNA against thesenucleases. This would increase the probability of nuclear translocationof intact plasmid leading to increase in the levels of gene expression.Since SSP has mostly amide bonds which connect individual amino acids aswell as the headgroup and steroid anchor, it would be stable enough forthe time period required for protecting and facilitating the pDNA intothe nucleus but would eventually degrade into biologically safemetabolites.

EXAMPLE 6 Cytotoxicity Assay

Cytotoxicity of SSP/p2CMVmIL-12 complexes prepared at different N/Pratios were assessed using an MTT assay. CT-26 colon adeno-carcinomacells were grown and maintained in RPMI 1640 medium (GIBCO-BRL,Gaithersburg, Md.), which was supplemented with 10% fetal bovine serum(FBS), 100 U/ml penicillin, 100 U/ml streptomycin and 50 μg/mlgentamycin (all from Gibco-BRL, Gaithersburg, Md.) at 37° C. andhumidified 5% CO₂.

Briefly, CT-26 cell lines were seeded in 96 well plates at 4000cells/well and incubated for 24 hours. After checking the cellconfluency, which was over 80%, SSP/p2CMVmIL-12 complexes prepared atN/P ratios ranging from 5/1 to 100/1 were added to the cells at a doseof 0.15 μg pmIL-12/well. Following 48 hrs of incubation, 25 μl of 5mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT,Sigma Chemical Co., St. Louis, Mo.) stock solution in phosphate bufferedsaline (PBS, Gibco-BRL) was poured into each well reaching a finalconcentration of 0.5 mg/ml MTT. The plate was then incubated at 37° C.in 5% CO₂ for 4 hrs. The medium was removed and 150 μl of dimethylsulfoxide (DMSO, Aldrich) was added to dissolve the formazan crystals.The plate was read spectrophotometrically at 570 nm in an ELISA platereader. The relative cell viability was calculated as[Abs]_(sample)/[Abs]_(control)×100.

SSP/p2CMVmIL-12 complexes prepared according to the procedure of Example5 were tested for cytotoxicity using MTT assay in CT-26 cells over awide range of N/P ratios. Commercially available polyamido amine (PAMAM)dendrimer (SuperFect)/p2CMVmIL-12 (6/1, w/w) complexes were used ascontrols for comparison. SuperFect, a G6 dendrimer has an approximatemolecular weight of 30,000 with a total of 140 protonatable amines.SSP/p2CMVmIL-12 complexes were indeed non-toxic to the cells, whenformulated at N/P ratio of 50/1 (+/−) and below (FIG. 6). In contrast,SuperFect/p2CMVmmIL-12 were toxic to the cells and the cell viability ofCT-26 cells was reduced to less than 60%. Cells treated withSuperfect/p2CMVmIL-12 complexes were granulated, and cell debris wasquite evident under light microscopy whereas the cell population wasunaffected and there was greater than 90% viability for all cellstreated with SSP/pDNA complexes up to N/P ratios as high as 50/1. SSPalone was also put to test for its cytotoxicity at various N/P ratiosand was found to be non-toxic at the amounts equivalent to those used inN/P ratios 20/1. However, the cell viability decreased to approx. 75% atN/P ratio 50/1.

EXAMPLE 7 In Vitro Transfections

SSP/pLuc and SSP/p2CMVmIL-12 complexes formulated at various N/P ratiosin 5% (w/v) glucose were evaluated for their transfection efficiency inCT-26 colon carcinoma cell lines. In the case of luciferase genecontrols, cells were lysed after transfection and the relative lightunits (RLU) and total protein concentration were determined. SSP/pLuccomplexes showed high transfection efficiencies between N/P ratios 20/1and 50/1. The transfection efficiency increased with the increase in N/Pratios but diminished slightly with ratios 50/1 and above which couldpossibly be due to cytotoxicity towards CT-26 cells. On visualinspection under light microscope some granulation of cells was found,and cytotoxicity tests indicate that above N/P ratios 50/1 the cellviability is reduced by about 80%, which would account for lower geneexpression. As shown in FIG. 7, the RLU values for SSP/pLuc complexesgave several orders of magnitude higher luciferase levels compared tonaked pLuc. Peptide sequence used as a control was not effective inmediating transfection and SSP alone did not give any gene expression.

In case of pLuc, CT-26 cells were seeded in 12-well tissue cultureplates at 1×10⁵ cells per/well in 10% FBS containing RPMI 1640 media.Cells achieved70% confluency within 24 hours, after which they weretransfected with SSP/pDNA complexes prepared at different N/P ratiosranging from 5/1 to 50/1 as described above. The total amount of pLucloaded was maintained constant at 1.5 μg/well and transfection wascarried out in the presence of the same serum-containing medium that wasused to maintain the cells. The cells were allowed to incubate in thepresence of complexes for 4 hours in CO₂ incubator followed byreplacement of 0.5 ml of RPMI 1640 containing 10% FBS. Thereafter thecells were incubated for additional 48 hours. Cells were lysed using1×lysis buffer (Promega, Madison, Wis.) after washing with cold PBS.Total protein assays were carried out using BCA protein assay kit(Pierce Chemical Co, Rockford, Ill.). Luciferase activity was measuredin terms of relative light units (RLU) using 96 well plate Luminometer(Dynex Technologies Inc, Chantilly, Va.). The luciferase activity wasmonitored and integrated over a period of 30 secs. The final values ofluciferase were reported in terms of RLU/mg total protein (FIG. 7). Inall the above experiments, naked pLuc and superfect as well as untreatedcultures were used as positive and negative controls, respectively.

In case of p2CMVmIL-12, all transfections were carried out exactly thesame way and after 48 hours of incubation the supernatants werecollected and stored at −20° C. until further use. Culture supernatantswere assayed for mIL-12 p70 using enzyme linked immunosorbent assay(ELISA) kits as suggested by the manufacturer.

In case of cytokine genes, the culture media was collected aftertransfection and the levels of mIL-12 p70 was determined using ELISA kitBDOptEIA™ for mIL-12 p70 (Pharmingen, San Diego, Calif.), which was usedaccording to the manufacturer's instructions. Briefly, ELISA plates(Nunc, Maxisorp, Denmark) were coated with capture antibody, sealed andkept overnight for antibody binding. The plate was washed several timesfollowed by incubation with assay diluent to block any non-specificbinding for one hour. After washing several times, the plate was thenincubated with samples and standards for 2 hours. After incubating withdetection antibody solution containing avidin-HRP reagent for 1 hour,the substrate solution was added to carry out enzymatic reaction. Thereaction was stopped by 2N H₂SO₄ and the plate was read at 450 nm usingBIO-RAD (model 3550) ELISA reader (Hercules, Calif.). The mIL-12 p70concentrations were reported in terms of pg/ml and were normalizedacross different samples.

Trends similar to luciferase transfection followed when a gradient ofvarious N/P ratios ≧20/1 was used. As shown in FIG. 8, the mIL-12 levelsfor SSP/pmIL-12 complexes were over 20 fold greater than nakedp2CMVmIL-12.

Therefore, soluble amphiphilic lipopeptides were synthesized that, inaddition to being completely biodegradable, result in high transfectionefficiencies without compromising survivability of cells.

1. A composition comprising a PRM2 peptide conjugated to a hydrophobicmoiety.
 2. The composition of claim 1 wherein said PRM2 peptidecomprises a peptide identified as SEQ ID NO:2.
 3. The composition ofclaim 1 wherein said hydrophobic moiety comprises a sterol.
 4. Thecomposition of claim 3 wherein said sterol is a member selected from thegroup consisting of cholestanol, coprostanol, cholesterol,epicholesterol, ergosterol, and ergocalciferol.
 5. The composition ofclaim 3 wherein said sterol comprises cholesterol.
 6. The composition ofclaim 1 wherein said hydrophobic moiety comprises a bile acid.
 7. Thecomposition of claim 6 wherein said bile acid is a member selected fromthe group consisting of cholic acid, deoxycholic acid, chenodeoxycholicacid, lithocholic acid, ursocholic acid, ursodeoxycholic acid,isoursodeoxycholic acid, lagodeoxycholic acid, glycocholic acid,taurocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid,dehydrocholic acid, hyocholic acid, and hyodeoxycholic acid.
 8. Thecomposition of claim 3 wherein said bile acid comprises lithocholicacid.
 9. The composition of claim 1 wherein said hydrophobic moietycomprises a fatty acid.
 10. The composition of claim 9 wherein saidfatty acid is a member selected from the group consisting of C4-C20alkanoic acids.
 11. The composition of claim 9 wherein said fatty acidis a member selected from the group consisting of butyric acid, valericacid, caproic acid, caprylic acid, capric acid, lauric acid, myristicacid, palmitic acid, and stearic acid.
 12. The composition of claim 1wherein said PRM2 peptide is conjugated to said hydrophobic moietythrough a peptide linkage.
 13. The composition of claim 12 wherein saidPRM2 peptide comprises a peptide identified as SEQ ID NO:2, and saidhydrophobic moiety comprises cholesterol.
 14. The composition of claim12 wherein said PRM2 peptide comprises a peptide identified as SEQ IDNO:2, and said hydrophobic moiety comprises lithocholic acid.
 15. Thecomposition of claim 1 wherein said hydrophobic moiety is conjugated tosaid PRM2 peptide through a non-terminal amino acid residue.
 16. Acomposition comprising a mixture of a nucleic acid and a conjugatecomprising a PRM2 peptide and a hydrophobic moiety.
 17. The compositionof claim 16 wherein said nucleic acid binds to said PRM2 peptide. 18.The composition of claim 16 wherein said nucleic acid comprises aplasmid.
 19. The composition of claim 16 wherein said PRM2 peptidecomprises a peptide identified as SEQ ID NO:2.
 20. The composition ofclaim 16 wherein said hydrophobic moiety comprises a sterol.
 21. Thecomposition of claim 20 wherein said sterol is a member selected fromthe group consisting of cholestanol, coprostanol, cholesterol,epicholesterol, ergosterol, and ergocalciferol.
 22. The composition ofclaim 20 wherein said sterol comprises cholesterol.
 23. The compositionof claim 16 wherein said hydrophobic moiety comprises a bile acid. 24.The composition of claim 23 wherein said bile acid is a member selectedfrom the group consisting of cholic acid, deoxycholic acid,chenodeoxycholic acid, lithocholic acid, ursocholic acid,ursodeoxycholic acid, isoursodeoxycholic acid, lagodeoxycholic acid,glycocholic acid, taurocholic acid, glycodeoxycholic acid,glycochenodeoxycholic acid, dehydrocholic acid, hyocholic acid, andhyodeoxycholic acid.
 25. The composition of claim 20 wherein said bileacid comprises lithocholic acid.
 26. The composition of claim 16 whereinsaid hydrophobic moiety comprises a fatty acid.
 27. The composition ofclaim 26 wherein said fatty acid is a member selected from the groupconsisting of C4-C20 alkanoic acids.
 28. The composition of claim 26wherein said fatty acid is a member selected from the group consistingof butyric acid, valeric acid, caproic acid, caprylic acid, capric acid,lauric acid, myristic acid, palmitic acid, and stearic acid.
 29. Thecomposition of claim 16 wherein said PRM2 peptide is conjugated to saidhydrophobic moiety through a peptide linkage.
 30. The composition ofclaim 29 wherein said PRM2 peptide comprises a peptide identified as SEQID NO:2, and said hydrophobic moiety comprises cholesterol.
 31. Thecomposition of claim 29 wherein said PRM2 peptide comprises a peptideidentified as SEQ ID NO:2, and said hydrophobic moiety compriseslithocholic acid.
 32. The composition of claim 16 wherein saidhydrophobic moiety is conjugated to said PRM2 peptide through anon-terminal amino acid residue.
 33. The composition of claim 16 whereinsaid nucleic acid encodes interleukin-12.
 34. The composition of claim33 wherein said nucleic acid comprises p2CMVmIL-12.
 35. A method fortransfecting a mammalian cell comprising contacting said cell with acomposition comprising a mixture of a nucleic acid and a conjugatecomprising a PRM2 peptide and a hydrophobic moiety, and then incubatingsaid cell under conditions suitable for growth thereof.
 36. The methodof claim 35 wherein said mammalian cell is a human cell.
 37. The methodof claim 35 wherein said nucleic acid binds to said PRM2 peptide. 38.The method of claim 35 wherein said nucleic acid comprises a plasmid.39. The method of claim 35 of claim 1 wherein said PRM2 peptidecomprises a peptide identified as SEQ ID NO:2.
 40. The method of claim35 wherein said hydrophobic moiety comprises a sterol.
 41. The method ofclaim 40 wherein said sterol is a member selected from the groupconsisting of cholestanol coprostanol cholesterol, epicholesterol,ergosterol, and ergocalciferol.
 42. The method of claim 40 wherein saidsterol comprises cholesterol.
 43. The method of claim 35 wherein saidhydrophobic moiety comprises a bile acid.
 44. The method of claim 43wherein said bile acid is a member selected from the group consisting ofcholic acid, deoxycholic acid, chenodeoxycholic acid, lithocholic acid,ursocholic acid, ursodeoxycholic acid, isoursodeoxycholic acid,lagodeoxycholic acid, glycocholic acid, taurocholic acid,glycodeoxycholic acid, glycochenodeoxycholic acid, dehydrocholic acid,hyocholic acid, and hyodeoxycholic acid.
 45. The method of claim 43wherein said bile acid comprises lithocholic acid.
 46. The method ofclaim 35 wherein said hydrophobic moiety comprises a fatty acid.
 47. Themethod of claim 46 wherein said fatty acid is a member selected from thegroup consisting of C4-C20 alkanoic acids.
 48. The method of claim 46wherein said fatty acid is a member selected from the group consistingof butyric acid, valeric acid, caproic acid, caprylic acid, capric acid,lauric acid, myristic acid, palmitic acid, and stearic acid.
 49. Themethod of claim 35 wherein said PRM2 peptide is conjugated to saidhydrophobic moiety through a peptide linkage.
 50. The method of claim 49wherein said PRM2 peptide comprises a peptide identified as SEQ ID NO:2,and said hydrophobic moiety comprises cholesterol.
 51. The method ofclaim 49 wherein said PRM2 peptide comprises a peptide identified as SEQID NO:2, and said hydrophobic moiety comprises lithocholic acid.
 52. Themethod of claim 35 wherein said hydrophobic moiety is conjugated to saidPRM2 peptide through a non-terminal amino acid residue.
 53. An plasmidconfigured for expressing p35 and p40 subunits of interleukin-12 undercontrol of at least one cytomegalovirus promoter.
 54. The plasmid ofclaim 53 wherein said plasmid is p2CMVmIL-12.
 55. A method for making anamphiphilic lipopeptide comprises conjugating a hydrophobic moiety to anon-terminal amino acid residue of a PRM2 peptide.